( B) RNAP II accumulation as shown by ChIP-Seq (purple) (40) with RNA-seq (+) (green) and (-) (tan) signal around the PAS. Reads were normalized as reads per base pair per gene. ( A) Metaplot of GRO-seq signal from TAIR10 annotated genes for Arabidopsis and human samples (8). For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 ☌ followed by 5 min at RT and consecutively washed three times before run-on.Īrabidopsis shows extensive 3′ RNAP accumulation in proximity to the PAS followed by rapid transcription termination. ( E) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. ( D) 5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 ☌ and 37 ☌ in T4 RNA ligase buffer. ( C) 32P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 ☌ and 20 ☌. ( B) Comparison of RppH activity on 32P-capped RNA in buffer NEB II vs. T4 RNAP synthesized RNA (264 nt) was kinased using T4 PNK and ATP or capped with the Vaccinia Capping System (M2080) and GTP, as described by the manufacturer. ( A) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). Sites are sorted based on the total GRO-seq signal observed within 400 bp of the intergenic peak. ( F) Intergenic sites were defined by DNase-seq peaks, and heat maps were generated ☑ kb from intergenic sites for signal from DNase-seq, GRO-seq, RNA-seq, H3K4me3, H3K9/27ac, and input in Arabidopsis, maize, and IMR-90 cells (,, –25). ( E) Metaplot of GRO-seq signal from annotated genes normalized for reads per bp per gene along y axis for Arabidopsis and human IMR-90 cells. ( D) Distribution of RNA-seq and GRO-seq reads relative to annotations or extended annotations (±500 bp) ( Right) for Arabidopsis and human IMR-90 cells. The Wilcoxon test was used to calculate P value. ( C) Ratio of nascent/steady-state transcript genome coverage as a function of GRO-seq/RNA-seq coverage for Arabidopsis seedlings and human IMR-90 cells (8). ( B) Browser shot of sample gene At4g10180.1 with normalized read densities along the y axis. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole.ĥ′GRO-seq GRO-seq RNA polymerase pausing nascent transcripts plant transcription. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved "TGT" core promoter motif and unreported transcription factor-binding sites. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5'GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles.
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